Composition comprising heparin as a non-thrombogenic surface coating agent

ABSTRACT

The present invention relates to a composition comprising heparin to be used as a non-thrombogenic surface when in contact with arterial blood flow. It also relates to a device treated on the surface thereof with such a composition.

This application is a 371 of PCT/NO99/00278, filed Sep. 9, 1999.

The present invention relates to a composition comprising heparin to beused as a non-thrombogenic surface coating agent when in contact witharterial blood flow. It also relates to a device treated on the surfacethereof with such a composition.

BACKGROUND OF THE INVENTION

Thrombosis is a major health problem in the industrialized world.Thrombosis related diseases cause the death of several millions ofpeople every year and the health care costs have been estimated to beover 90 billion US dollars just for the USA.

There are two different kinds of thrombosis, arterial and venous. As thenames suggest the arterial thrombosis occurs in the arteries and thevenous thrombosis occurs in the veins. The arterial thrombus is formedat high flow rates and platelets are the main component. Platelets aresmall cells, diameter around 2 μm, circulating in the blood. Their mainfunction is to participate in the haemostasis. When platelets areexposed to collagen in damaged or altered vessel walls or in wounds orwhen exposed to foreign surfaces they adhere to the surfaces, becomeactivated and start to form aggregates. When this occurs in an artery athrombus with aggregated platelets is formed. The arterial thrombus hasalso been called the white thrombus because of its appearance which isdue to the fact that it mainly contains platelets and very fewerythrocytes (red blood cells).

The venous thrombus is formed at low flow rates and the bloodcoagulation system is the major participant. In plasma a number ofproenzymes and effector proteins are present that together constitutethe coagulation system. The system can be triggered in several ways andin a cascade-like process one enzyme activates a proenzyme and theformed enzyme activates the next proenzyme. The final enzyme formed isthrombin and that cleaves off two peptides from the plasma proteinfibrinogen, which then results in a rapid aggregation of the modifiedfibrinogen to form a gel, and a fibrin clot or thrombus is formed. Thevenous thrombus is called the red thrombus because it containserythrocytes embedded in fibrin.

Diseases related to arterial thrombosis are: myocardial infarction,thrombotic stroke, and peripheral arterial disease. In myocardialinfarction a thrombus is formed in one of the coronary arteries and theblood supply to the corresponding part of the heart muscle is stopped orstrongly diminished resulting in death of that part of the heart muscle.In thrombotic stroke the blood flow in a cerebral artery is blocked by athrombus, which usually has been formed somewhere else in thecirculation and followed the blood flow to the brain. As the brain isvery sensitive to oxygen deprivation, the part of the brain supplied byblood from this artery will be damaged.

Myocardial infarction and thrombotic stroke are very serious conditionswith high mortality and therefore, many treatment efforts today intendto prevent them from occurring. The mostly used drug is aspirin(acetylsalicylic acid), which inhibits the activation and aggregation ofplatelets and thereby prevents the arterial thrombus formation. Largeclinical studies have shown that one aspirin a day significantly reducesthe risk for having myocardial infarction. For prevention of thromboticstroke there is also another drug which is commonly used. That isTiclopedin, which also inhibits platelet aggregation but throughsomewhat different mechanisms than aspirin.

Diseases related to venous thrombosis are deep venous thrombosis (DVT)and pulmonary embolism. In deep venous thrombosis a thrombus is formedin one of the veins in the extremities usually the legs. That thrombusdiminishes the return flow of blood and results in diminished bloodsupply to that part of the leg or arm. The leg or arm becomes swollenand painful. The condition as such is not life-threatening but if nottreated the thrombus can grow and extend and pieces can leave by thereturn flow and get stuck in the lungs. That condition which islife-threatening is called pulmonary embolism. The clinical practice indealing with venous thrombosis can be divided into prevention andtreatment. However, the drugs used are the same—the difference is in thedosing and time of treatment.

The drugs used are heparin, low molecular weight heparin and dicoumarolderivatives. All of them act as to diminish coagulation and fibrinformation, which is the key process in venous thrombosis. Heparin is asulfate-containing polysaccharide, which on large scale is isolated fromintestinal mucus of swine. It has for many decades been used clinicallyas an agent for the treatment and prevention of venous thrombosis.Heparin is heterogeneous with a molecular weight from 5,000 to 30,000daltons and with an average molecular weight of about 12,000-15,000daltons. Heparin and low molecular weight heparin exert anticoagulantactivity by drastically increasing the rate whereby the physiologicalcoagulation inhibitor, antithrombin III (AT), inactivates activatedcoagulation factors. Only about one third of heparin molecules do bindAT and have a strong anticoagulant activity. This is related to the factthat they contain a specific pentasaccharide sequence with a strongaffinity for AT. This fraction of heparin is called the high affinity orHA-fraction. The residual part, the low affinity (LA) fraction, isessentially devoid of anticoagulant activity. Regarding the moreimportant thing, the in vivo antithrombotic activity, the situation ismore complex, as not only activation of AT is important but also othermechanisms, such as release of Tissue Factor Pathway Inhibitor (TFPI)contribute to the antithrombotic effect. LA-heparin releases TFPI andthus contributes to the antithrombotic effect of whole heparin despitehaving no anticoagulant activity.

Heparin also affects platelets but it acts as a weak stimulator ofplatelet aggregation and it also potentiates the platelet aggregatoryaction of adenosine diphosphate. There is no difference between the HA-and LA-heparin regarding their ability to affect platelets in thisrespect as shown by Holmer et al., Thromb Res 1980; 18: 861-69.

Dicoumarol derivatives have antithrombotic action through diminishingsynthesis of some coagulation factors in their biologically activeforms. That process takes some time and that is why the dicoumarolderivatives cannot be used for immediate antithrombotic treatment.

During the last two decades there has been a large progress in thedevelopment of devices for various types of implantation or use inmachines, where there is contact with blood. However, that has alsocreated a new type of thrombosis problem. When blood comes into contactwith other materials than the fresh natural wall of the blood vessel,activation of the coagulation system starts to occur and thrombotisationcan follow. The thromboses formed are of the arterial type withplatelets as a dominating element when the foreign surface is subjectedto arterial blood flow (Hanson et al. Biomaterials 1982; 519-30). Invenous blood flow the situation is mixed with both platelets andcoagulation involved.

To prevent thrombosis in devices it is possible to use antiplatelet andantithrombotic drugs. This is not an ideal solution however, as thatimposes a bleeding risk and further drug treatment often has to go onfor long times which is a disadvantage. That is why far going effortshave been made to find materials of reduced tendency to form thrombosis.Various polymers and plastic materials have been tried. Thehydrophilicity/hydrofobicity of the surface has been varied but nobreak-through composition has been found. As platelets are negativelycharged at physiological pH, studies have been performed with surfacescontaining negative charges, where it could be expected that theadhesion would be diminished due to the electrostatic repulsion. So farthe most successful example of making a surface less thrombogenicthrough coating of the surface with a negatively charged polymer is touse heparin for the coating to inhibit platelet adhesion. Heparin hasthe advantage compared to synthetic polymers that it is a physiologicalcompound. It is also the most strongly negatively charged molecule thatcan be found in the human body. There is no difference between HA- andLA-heparin in this respect.

Different technologies have been developed to attach heparin to surfacesin order to make them less thrombogenic. Ionic binding of heparin topolycationic surfaces has been tried but has been less successful asheparin leached from the surface resulting in loss of antithromboticproperties.

One of the most successful processes for rendering a medical devicenon-thrombogenic is achieved through covalently binding a heparinfragment to a modified surface of the medical device. The general methodand improvements thereof are described in the following Europeanpatents: EP-B-0086186, EP-B-0086187 and EP-B-0495820.

These patents describe the preparation of surface modificationsubstrates which are achieved through firstly, a selective cleavage ofthe heparin polysaccharide chain while aldehyde groups are introducedthrough an oxidation with nitrous acid. Secondly an introduction of oneor more surface modifying layers carrying amino groups on the surface ofthe medical device, and thereafter the aldehyde groups on thepolysaccharide chain are reacted with primary amino groups on surfacemodifying layers followed by a reduction of the intermediate Schiff'sbases to stable secondary amine bonds with for instancecyanoborohydride.

This technology has made it possible to prepare stable and well-definedantithrombogenic surface modifications for medical devices.

There are known many other surface modifications which claim to achievesimilar or even better results, such as for instance described inEP-A-0200295 (U.S. Pat. No. 4.600,652. U.S. Pat. No. 6,642,242.) basedon substrates having a layer of a polyurethane urea to which heparinmodified to contain aldehyde groups through an oxidation with nitrousacid or periodate, may be bound by covalent links.

Another antithrombogenic surface modification which may be mentioned isdescribed in EP-B-0309473. The surface of the device is modified throughthe coating with a layer of lysozyme or a derivative thereof to whichheparin is adhered.

Yet another surface modification for producing antithrombogenic articlesis described in U.S. Pat. No. 4,326,532. In this case, the layeredantithrombogenic surface comprises a polymeric substrate, a chitosanbonded to the polymeric substrate and an antithrombogenic agent bondedto the chitosan coating. Japanese Patent Laid-Open No. 04-92673 relatesto an antithrombogenic hemofilter also using a chitosan layer forbinding heparin. The listing of prior art processes for preparingantithrombogenic surfaces is by no means complete, and it is a clearindication that it is difficult to prepare such coated medical articleswhich exhibit the properties necessary of successful use in patients,namely stability of the coating, no adverse change of properties of thesubstrate to be coated and sufficiently high and long lastingantithrombogenic activity.

Thus even if coating of surfaces with heparin has been successful inreducing thrombogenicity there is still need for improvement and the useof a fraction of heparin having optimal properties could be an importantstep forward.

The present invention describes the preparation and characterization ofHA- and LA-heparin, intended for coating of surfaces, as obtained byaffinity chromatography on matrix bound AT. It further deals withcoating of surfaces of devices with the HA- and LA-heparin preparations.Separation of HA- and LA-fractions of heparin followed by coupling tosurface has previously been performed by Yuan et al. (J Biomed Mater Res1993; 27: 811-20 and J Appl Biomater 1995; 6: 259-66). In the study ofYuan et al. (1995) it was found that the LA-heparin coated surface hadhigher anticoagulant activity, measured as anti Factor Xa activity, thanthe HA-heparin coated surface.

In the present invention a surface coated vascular implant (stent) wasinvestigated in an animal experimental thrombosis model. The animalmodel used was an arterio-venous shunt model in baboons. In that modelthe blood from one artery passes through a tubing containing thematerial to be studied and then to a vein. The thromboses formed in thismodel are of the arterial type and the thrombus formation is followed bymeasuring the accumulation of radiolabelled platelets.

Contrary to what could be expected from what is known on mechanisms ofarterial thrombosis, with its predominant platelet involvement, and whatis known about effects of HA- and LA-heparin on platelets, it was foundthat, when coupled to surface, the HA-fraction was much more efficientthat the LA-fraction to prevent formation of arterial thrombosis.

Through the present invention improved non-thrombogenic activity can beobtained and as a result thereof the following advantages can beachieved:

sufficient non-thrombogenicity can be obtained with lower quantities ofimmobilized heparin. This is especially important for material anddevices associated with difficulties to immobilize large amounts ofheparin.

higher non-thrombogenicity can be obtained with the same amount ofimmobilized heparin. This is especially important in applications withstrong thrombogenic stimuli e.g. in situations with low blood flow andapplications like catheters and vascular grafts with narrow lumen or incases where the patient otherwise would require additional systemicheparinization.

SUMMARY OF THE INVENTION

One object of the invention is to provide a composition comprisingheparin to be used as a non-thrombogenic surface coating when in contactwith arterial blood flow. Another object of the invention is to providea device treated on the surface thereof with such a composition.

DETAILED DESCRIPTION OF THE INVENTION

One object on the invention is to provide a composition comprisingheparin enriched with respect to high affinity, HA, heparin, to becoupled to a surface of a medical device, optionally together with asuitable carrier.

Preferably, the composition comprises heparin purified with respect tohigh affinity, HA, heparin.

The composition according to the invention is optionally combined with acarrier to effect the immobilization of the heparin onto the surface ofthe medical device.

The carrier is selected from the group of organic compounds carryingfunctional groups such as amino, aldehydo, hydroxyl, carboxylic acid,carbodiimido or other reactive functional groups that can be bound tofunctional groups present in or introduced into the heparin.

The functional compounds could be low molecular weight compounds orpolymers. Examples of low molecular weight compounds carrying functionalgroups are tridodecylmethyl-ammonium chloride, benzalkonium chloridederivatives, ethyl dimethylaminopropyl carbodiumide and glutaraldehyde.Examples of polymers are e.g. polyamines such as polyethylenimine (PEI)or polylysine, polycarboxylic acids like polyacrylic acid, polyalcoholslike polyvinyl alcohol or polysaccharides and other functional polymersor combinations thereof. The functional groups are present in such anextent that a sufficient amount of HA-heparin can be bound and a strong,antithrombogenic activity can be obtained.

Another object of the invention is to provide a device treated on thesurface thereof with a composition according to the invention. Asuitable device is selected from but not limited to the group consistingof stents, grafts, stent-grafts, catheters, heart-valves, filters,tubings and membrane containing devices.

Pig mucosal heparin was depolymerized by nitrous acid as described inLarm et al. EP-86186B1. The resulting depolymerized heparin wasfractionated by affinity chromatography on matrix bound antithrombin IIIinto its HA- and LA-fractions (Andersson LO et al. Tromb Res 1979; 9:575-83). As expected the HA-fraction showed very high anticoagulantactivity whereas the LA-fraction had very low activity. Two groups ofstents of stainless steel (Palmaz-Schatz, PS153, Cordis, Warren, N.J.USA) were then coated (Larrn et al. EP-86186B1) with the two fractions,respectively. Stents are a kind of tublar nets that are used to supportthe vascular walls and they are used in connection with invasivecardiovascular procedures. Determination of degree of heparin bindingshowed that essentially the same amounts of the HA-fraction the andLA-fraction, respectively, had been coupled to the surfaces of the twogroups of stents. To evaluate the antithrombogenic properties of the twosurfaces an establish animal model was used. The model used was anarterio-venous shunt model in baboons. In that model the blood from oneartery passes through a tubing containing the material to be studied andthen to a vein. The thromboses formed in this model are of the arterialtype and the thrombus formation is followed by measuring theaccumulation of radiolabelled platelets (Cadroy Y et al. J Lab Clin Med1989; 113: 436-48).

Stents were placed and expanded in the ex-vivo arterio-venous shunt in anonanticoagulated baboon that had been injected with radiolabelledplatelets. Accumulation of platelets to the stent surface wasintermittently monitored by a gamma camera and recorded for a two hourperiod. Non-coated stents were used as controls. In the control stentsplatelets immediately started to adhere and continued to accumulate forthe duration of the experiment. Stents coated with the LA-heparinfraction showed initially significantly less platelet accumulation thanthe control but after 40 minutes accumulation started to occur and aftertwo hours there was as much platelet as with the control stents. Incontrast no platelet accumulation occurred on the stents coated with theHA-heparin fraction. Even after two hours there was no sign of plateletaccumulation. Thus the stents coated with HA-heparin fraction havesuperior non-thrombogenic properties.

As mentioned above coating of surfaces with heparin has been fairlysuccessful in reducing thrombogenicity. However, there is still need forimprovement and as the HA-fraction of heparin seem to be responsible forthe main part of the antithrombotic effect it would be an advantage tohave a surface enriched with respect to that fraction.

In this study stents have been chosen as the type of device andartificial surface to be investigated. The reason being that the stentis well suited for evaluation in this animal model for arterialthrombosis. However, the type of device whether it is catheters,filters, tubings, vascular grafts or stents is not important for thethrombogenicity of the surface. That is determined by the properties ofthe surface rather than by the device itself or its bulk material. Thusthe conclusions regarding non-thrombogenicity of surfaces reached forthe stents are not limited to stents but covers all types of artificialdevice surfaces in contact with arterial blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the adhesion of platelets to stents coatedwith the HA- and LA-heparin fractions, respectively, and to controlstents. Uncoated PS153 stents were used as controls. The numbers inparentheses relate to the number of experiments and animals used inevery case.

MATERIALS AND METHODS

Pig mucosal heparin was depolymerized by nitrous acid as described inLarm et al. EP-0086186B1. The resulting depolymerized heparin wasfractionated by affinity chromatography on matrix bound AT into its HA-and LA-fractions (Andersson LO et al. Tromb Res 1976; 9: 575-83). HumanAT was obtained from Pharmacia & Upjohn, Stockholm. Activated CHSepharose 4B was obtained from Pharmacia Biotech AB, Uppsala, Sweden.Coronary stents, Palmaz-Schatz, PS 153, were supplied by Cordis, Warren,USA. Chromogenic substrates S-2238 and S-2765 were from Chromogenix AB,Mölndal, Sweden. The 4th International Standard for Heparin activity wasobtained from the National Institute for Biological Standard andControl, Hertfordshire, United Kingdom.

Preparation of AT-Sepharose was performed according to gel manufacturersinstructions. The lyophilized gel (125 g) was reconstituted in bufferand was then reacted with 4 g of AT. The resulting AT-Sepharose (400 ml)had the capacity to bind about 100 mg of HA-heparin. The anticoagulantactivities of heparin fractions were determined by a thrombin inhibitionassay and by a Factor Xa inhibition assay essentially according theEuropean Pharmacopoeia methods for Low Molecular Mass Heparin using thechromogenic substrates S-2238 and S-2765, respectively. Standard curveswere constructed using the 4th International Standard for Heparin andthe specific activities were expressed in international units per mg(IU/mg). The heparin content of the heparin subfractions was determinedby the carbazole-H₂SO₄ method. The amount of heparin bound to thesurfaces (heparin density) was determined by a chemical method andexpressed as amount of heparin per unit surface area (μg/cm²).

The invention is illuminated by the following Examples. These Examplesare only illustrative and are not intended to limit the invention in anyway whatsoever.

All publications mentioned herein are hereby incorporated by reference.By the expression “comprising” we understand including but not limitedto. Thus, other nonmentioned substances or additives may be present.

EXAMPLES Example 1

Partially nitrous acid depolymerized heparin was separated into its HA-and LA-fractions by affinity chromatography on AT-Sepharose essentiallyaccording to Andersson et al. 1976. Heparin loads of 200 mg in 4 ml of0.15M NaCl were applied to the column and eluted by 500 ml of 0.15M NaClfollowed by 500 ml of 2.0M NaCl. The eluate was collected in tubes in 10ml portions and their heparin content was analyzed by the carbazolemethod. Tubes containing the LA- and HA-heparin respectively werecollected. Each run yielded about 140 mg LA- and 50 mg HA-heparin. Theresulting HA- and LA-fractions were characterized with respect toanticoagulant activity. Results are shown in Table 1.

The HA-fraction expressed high anticoagulant activities, 344 IU/mg and318 IU/mg, in the thrombin- and factor Xa inhibition assays,respectively. The LA-fraction was essentially devoid of activity (<5IU/mg) by both assays (Table 1).

EXAMPLE 2

Coronary stents were coated with the heparin fractions using technologyessentially as previously described (Larm et al. EP-86186B1). Twobatches of fifty stents were coated with the HA-fraction and theLA-fraction, respectively. The coated stents were then sterilized usingethylene oxide (EO). The heparin density of the stents is shown in Table2. The heparin density was essentially the same, about 5 μg/cm², for thetwo preparations.

The non-thrombogenic properties of the two different heparin coatings onstents were studied in a primate animal experimental model. Stents wereplaced and expanded in an ex-vivo AV shunt in a non-anticoagulatedbaboon that had been injected with ^([1])In radiolabelled platelets.Adhesion of platelets to the stent surface was intermittently monitoredby a gamma camera and recorded for a 2 h period. Non-coated PS 153stents were used as controls. The results are presented in FIG. 1. Noplatelet adhesion was seen for the HA-stents. In contrast, plateletsimmediately started to adhere upon implantation of the non-coatedcontrol-stent and adhesion continued during the time period studied. TheLA-stents showed slightly less adhesion than the control stent up to 1hour but after 2 hours this difference was abolished and the LA-stentbehaved essentially as the control stents.

TABLE 1 Anticoagulant activities of HA- and LA-fractions of partiallydepolymerized heparin Activity Activity thrombin inhibi- Factor Xa inhi-Preparation Recovery % tion IU/mg bition IU/mg HA 27 344 318 LA 67 <5 <5

TABLE 2 Heparin density on PS153 stents coated with partly depolymerizedheparin and its HA- and LA-fractions. Heparin density Preparation μg/cm²HA 4.5 LA 4.3

What is claimed is:
 1. A method of use for preparing a non-thrombogenicsurface for preventing formation of arterial thrombosis comprising:using heparin enriched with respect to high affinity, HA, heparin.
 2. Amethod of use for preparing a non-thrombogenic surface for preventingformation of arterial thrombosis comprising: using heparin enriched withrespect to high affinity, HA, heparin, coupled to a surface.
 3. A methodof use for preparing a non-thrombogenic surface for preventing formationof arterial thrombosis comprising: using a composition containingheparin enriched with respect to high affinity, HA, heparin, coupled toa surface, together with a suitable carrier.
 4. A method of useaccording to claim 1, wherein the high affinity, HA, heparin ispurified.
 5. A method of use according to claim 1, wherein the highaffinity, HA, heparin is coupled to a surface by end point attachment.6. A method of use according to claim 3, wherein the carrier is selectedfrom the group of organic compounds having functional groups such asamino, aldehydo, hydroxyl, carboxylic acid, carbodiimido or otherreactive functional groups that can be bound to functional groupspresent in or introduced into the heparin, and wherein the functionalcompounds can be low molecular weight compounds or polymers.
 7. A methodof use according to claim 6, wherein the low molecular weight compoundsor polymers are selected from the group of tridodecylmethylammoniumchloride, benzalkonium chloride derivatives, ethyl dimethylaminopropylcarbodiimide, glutaraldehyde, polyamines, polyethylenimine (PEI),polylysine, polycarboxylic acids, polyacrylic acid, polyalcohols,polyvinyl alcohol, polysaccharides and combinations thereof.
 8. A methodof use according to claim 1, further comprising: treating a surface of adevice with a composition comprising said heparin enriched with highaffinity, HA, heparin.
 9. A method of use according to claim 8, whereinthe device is selected from the group consisting of stents, grafts,stent-grafts, catheters, heart-valves, filters, tubings and membranecontaining devices.